ES2531551T3 - Methods for building genetic package presentation libraries for members of a diverse peptide family - Google Patents

Abstract

A method of obtaining a library that has a diverse family of peptides, polypeptides or proteins on the surface of a genetic package, the method comprising the steps of: (i) cleaving a nucleic acid at a desired location by a method comprising the steps of: (a) contacting a single stranded nucleic acid with a single stranded oligonucleotide, the single stranded oligonucleotide being complementary to the single stranded nucleic acid in the region in which the cleavage is desired; wherein the single stranded nucleic acid and the single stranded oligonucleotide are associated to form a locally double stranded region of the single stranded nucleic acid, in which the locally double stranded region comprises a restriction endonuclease recognition site; and (b) cleaving the nucleic acid at the restriction endonuclease recognition site, in which the cleavage comprises contacting a restriction endonuclease with the locally double-stranded region, in which the restriction endonuclease is specific to the recognition site of restriction endonuclease; the contacting and cleavage steps being carried out at a temperature between 45 ° C and 75 ° C, in which the single stranded nucleic acid and the single stranded oligonucleotide are associated to form a locally double stranded region of the single stranded nucleic acid, in which the rest of the single stranded nucleic acid it is single stranded, and in which the restriction endonuclease is active at temperature; and (ii) presenting a member of the family of peptides, polypeptides or proteins encoded by the nucleic acid cleaved on the surface of a genetic package and collectively presenting at least a portion of the family's diversity.

Description

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Another example of selecting an appropriate restriction endonuclease recognition site involves FR1 cleavage of the human lambda light chain. Table 400 shows the 31 known human lambda FR1 germline gene sequences. Table 405 shows restriction endonuclease recognition sites found in human FR1 lambda germline genes. HinfI and DdeI are the most preferred restriction endonucleases for cutting human lambda chains in FR1.

After the site or sites appropriate for cleavage are selected, one or more short oligonucleotides are prepared to functionally complement, alone or in combination, the selected recognition site. Oligonucleotides also include sequences that flank the recognition site in most amplified genes. This flanking region allows the sequence to hybridize with the single stranded DNA sufficiently to allow cleavage by the specific restriction endonuclease of the chosen site.

The actual length and sequence of the oligonucleotide depends on the recognition site and the conditions used for contacting and excision. The length must be sufficient for the oligonucleotide to be functionally complementary to the single stranded DNA along a region large enough to allow the two strands to associate, so that cleavage occurs at the chosen temperature and only at the desired location.

Typically, the oligonucleotides of this preferred method of the invention are about 17 to about 30 nucleotides in length. Below about 17 bases, hybridization is too weak, and above 30 bases there may be a loss of specificity. A preferred length is 18 to 24 bases.

Oligonucleotides of this length need not be identical complements of the germline genes. Instead, a few wrong pairings taken can be tolerated. Preferably, however, no more than 1-3 erroneous pairings are allowed. Some mismatches do not adversely affect the hybridization of the oligonucleotide with single stranded DNA. Therefore, it is said that the two DNAs are functionally complementary.

The second method for manipulating the amplified single stranded DNAs of this invention for cloning comprises the steps of:

(i)

contacting the single stranded nucleic acid with a partially double stranded oligonucleotide, the single stranded region of the oligonucleotide complementary to the single stranded nucleic acid in the region where cleavage is desired, and the double stranded region of the oligonucleotide having a restriction endonuclease recognition site Type II-S, whose cleavage site is located at a known distance from the recognition site; Y

(ii)

cleaving the nucleic acid only at the cleavage site formed by the complementation of the nucleic acid and the single stranded region of the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the selected temperature.

This second method uses universal restriction endonucleases ("ERU"). ERUs are partially double stranded oligonucleotides. The single-stranded or overlapping portion of the ERU consists of a DNA adapter that is functionally complementary to the sequence to be cleaved into the single-stranded DNA. The double stranded portion consists of a type II-S restriction endonuclease recognition site.

The ERU method of this invention is specific and precise, and can tolerate some (for example, 1-3) erroneous pairings in the complementary regions, that is, it is functionally complementary to that region. In addition, the conditions under which the ERU is used can be adjusted, so that most of the genes that are amplified can be cut, reducing the preference in the library produced from those genes.

The sequence of the single-stranded DNA adapter or overlapping portion of the ERU typically consists of about 14-22 bases. However, longer or shorter adapters can be used. The size depends on the ability of the adapter to associate with its functional complement in the single-stranded DNA and the temperature used to contact the ERU and single-stranded DNA at the temperature used to cleave the DNA with the type II-S enzyme. The adapter must be functionally complementary to the single stranded DNA along a region large enough to allow the two strands to associate so that cleavage can occur at the selected temperature and at the desired location. Single or overlapping portions 14-17 bases in length, and more preferably 18-20 bases in length, are preferred.

The site selected for cleavage using the ERU is preferably one that is conserved substantially in the amplified DNA family. In comparison to the first cleavage method of this invention, these sites need not be endonuclease recognition sites. However, like the first method, the sites

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The single stranded portion of the adapter is complementary to the cleavage region in the single stranded DNA. The overlap can be from about 2 bases to about 15 bases. The longer the overlap, the more effective the ligation will probably be. A preferred length for overlap is 7 to 10. This allows some erroneous pairings in the region, so that diversity can be captured in this region.

The single-stranded region or overlap of the partially double-stranded adapter is advantageous because it allows cleaved DNA to be captured at the selected site, but not other fragments. Such fragments would contaminate the library with genes encoding sequences that will not fold into appropriate antibodies and are probably non-specific adhesives.

An illustration of the use of a partially double-stranded adapter in the methods of this invention involves linking said adapter with a human FR3 region that has been cleaved, as described above, to 5’-ACnGT3 ’using HpyCH4lll, Bst4CI or Taal.

Table 250 F.2 shows the lower strand of the double stranded portion of the ligation adapter with the stranded lower stranded DNA. Since the HpyCH4lll site is so far to the right (as shown in Table 206), a sequence can be added that includes the AflII site as well as the XbaI. This lower strand portion of the partially double-stranded adapter, H43.XAExt, incorporates both XbaI and AflII sites. The upper strand of the double-stranded portion of the adapter has no place (due to erroneous pairings planned in the segments opposite the XbaI and AflII sites of H43.XAExt), but will hybridize very closely with H43.XAExt. H43AExt contains only the AflII site and will be used with the upper strands H43.ABr1 and H43.ABr2 (which have intentional alterations to destroy the AflII site).

After ligation, the desired captured DNA can be amplified again by PCR, if desired, using in the preferred embodiment a primer for the 3 'constant region of the antibody gene, and a primer for part of the double stranded region of the antibody. adapter. Primers can also carry restriction endonuclease sites for use in the cloning of amplified DNA.

After ligation, and perhaps amplification, of the partially double-stranded adapter with the single-stranded amplified DNA, the composite DNA is cleaved at selected 5 'and 3' endonuclease recognition sites.

The cleavage sites useful for cloning depend on the phage or phagemid in which the cassette will be inserted, and the sites available in the antibody genes. Table 1 provides restriction endonuclease data for 75 human light chains. Table 2 shows corresponding data for 79 human heavy chains. In each Table, endonucleases are ordered by increasing cutoff frequency. In these Tables, Nch is the number of chains cut by the enzyme, and Ns is the number of sites (some chains have more than one site).

From this analysis, Sfll, NotI, AflII, ApaLI, and Ascl are very suitable. Sfil and NotI are preferably used in pCES1 to insert the heavy chain display segment. ApaLI, and Ascl are preferably used in pCES1 to insert the light chain display segment.

BstEII sites appear in 97% of JH germline genes. In rearranged V genes, only 54/79 (68%) of the heavy chain genes contain a BstEII site, and 7/61 of these contain two sites. Thus, 47/79 (59%) contain a single BstEII site. An alternative to the use of BstEII is to split the end of JH by ERU and bind it to a synthetic oligonucleotide encoding part of CH1.

An example of preparing a family of DNA sequences using the methods of this invention involves capturing diversity of human CDR 3. As described above, the mRNAs of various autoimmune patients are reverse transcribed into lower strand cDNA. After the upper strand RNA is degraded, the lower strand is immobilized and a short oligonucleotide is used to cleave the cDNA in the 5 'direction of CDR 3. Then a partially double stranded synthetic DNA adapter is hybridized with the DNA, and the DNA is amplified using a primer for the adapter and a primer for the constant region (after FR4). The DNA is then cleaved using BstEII (in FR4) and an appropriate restriction endonuclease for the partially double-stranded adapter (for example Xba I and Aflll (in FR3)). The DNA is then ligated into a synthetic VH skeleton such as 3-23.

An example of preparation of a single stranded DNA that was cleaved using the ERU method involves the human kappa chain. The cleavage site in the sense strand of this chain is represented in Table 512. The kapextURE oligonucleotide hybridizes with the oligonucleotides (kaBR01UR, kaBR02UR, kaBR03UR and kaBR04UR) to form a partially double-stranded DNA. This DNA is then ligated with the cleaved soluble kappa chains. The ligation product is then amplified using kapextUREPCR and CKForeAsc primers (which inserts an AscI site after the C kappa end). This product is then cleaved with ApaLI and Ascl and ligated with similarly cut receptor vector.

Another example involves the cleavage illustrated in Table 515. After excision, an extender (ON_LamEx133) and four bridge oligonucleotides (ON_LamB1-133, ON_LamB2-133, ON_LamB3-133 and ON_LamB4-133) are hybridized to form a partially double-stranded DNA . That DNA binds with the strand sense strands

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The soluble PCR product was processed on a gel and showed a band of approximately 700 n, as expected (FIGURES 5 and 6). The DNA was cleaved with ApaLI and AscI enzymes, gel purified and ligated with similarly cleaved pCES1 vector. The presence of the correct size insert was checked by PCR in several clones as shown in FIGURE 15.

Table 500 shows the DNA sequence of a kappa light chain captured by this procedure. Table 501 shows a second sequence captured by this procedure. The closest bridge sequence was complementary to the 5’-agccacc-3 ’sequence, but the captured sequence is 5’-Tgccacc-3’, which shows that some mismatch is tolerated in the overlapping region.

Example 2: Diversity Construction of Synthetic CDR1 and CDR2 in V-3-23 VH Frame

A diversity of Synthetic Complementarity Determination Region (CDR) 1 and 2 in the VH 3-23 framework was constructed in a two-stage procedure: first, a vector containing the VH 323 framework was constructed, and then assembled a synthetic 1 and 2 CDR and was cloned in this vector.

For the construction of the V3-23 framework, 8 oligonucleotides and two PCR primers were designed (long oligonucleotides: TOPFR1A, BOTFR1B, BOTFR2, BOTFR3, F06, BOTFR4, ON-vgC1, and ON-vgC2 and primers: SFPRMET and BOTPCRPRIM, shown in Table 600) that overlap, based on the Genebank sequence of VH V323. The design incorporated at least one useful restriction site in each region of the framework, as shown in Table 600. In Table 600, the segments that were synthesized are shown in bold, overlapping regions are underlined, and priming regions PCR at each end are underlined. A mixture of these 8 oligonucleotides was combined at a final concentration of 2.5 uM in a Polymerase Chain Reaction (PCR) of 20 ul. The PCR mixture contained 200 µM dNTP, 2.5 mM MgCl2, 0.02 U Pfu Turbo ™ Polymerase DNA, Qqgen 1U Taq HotStart Polymerase and 1X Qiagen PCR buffer. The PCR program consisted of 10 cycles of 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 30 seconds. The assembled DNA sequence V3-23 was then amplified, using 2.5 ul of a 10-fold dilution of the initial PCR in 100 ul PCR reaction. The PCR reaction contained 200 µM dNTP, 2.5 mM MgCI2, 0.02 U Pfu Turbo ™ DNA Polymerase, 1Q Taq HotStart Polymerase DNA, 1X Qiagen PCR buffer, and 2 external primers (SFPRMET and BOTPCRPRIM) at a concentration of 1 uM. The PCR program consisted of 23 cycles at 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 60 seconds. The VH V3-23 DNA sequence was digested and cloned into pCESI (phagemid vector) using the Sfil and BstEII restriction endonuclease sites (All restriction enzymes mentioned herein were provided by New England BioLabs, Beverly, MA, and were used according to the manufacturer's instructions).

Filler sequences (shown in Table 610 and Table 620) were introduced into pCES1 to replace CDR1 / CDR2 sequences (900 bases between ER BspEI and XbaI sites) and CDR3 sequences (358 bases between AflII and BstEII), before clone the diversity of CDR1 / CDR2. The new vector is pCES5, and its sequence is given in Table 620. Having fillings in place of the CDRs avoids the risk of a parental sequence being overrepresented in the library. The CDR1-2 filler contains restriction sites for BglII, Bsu36I, BclI, XcmI, MluI, PvuII, HpaI, and HincII, the unique underlining sites being within the pCES5 vector. The filler that replaces CDR3 contains the unique restriction endonuclease site RsrII. Filler sequences are fragments of the E. coli penicillase gene.

For the construction of the diversity of CDR1 and CDR2, 4 overlapping oligonucleotides (ON-vgC1, ON_Br12, ON_CD2Xba and ON-vgC2, shown in Table 600 and Table 630) that encode CDR1 / 2, plus flanking regions were designed. A mixture of those 4 oligonucleotides was combined at a final concentration of 2.5 µM in a 40 ul PCR reaction. Two of the 4 oligonucleotides contained variegated sequences positioned in CDR1 and CDR2. The PCR mixture contained 200 µM dNTP, 2.5 U Pwo DNA Polymerase (Roche) and 1X Pwo PCR buffer with 2 mM MgSO4. The PCR program consisted of 10 cycles at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 60 seconds. This assembled CDR1 / 2 DNA sequence was amplified, using 2.5 ul of the 100 ul PCR reaction mixture. The PCR reaction contained 200 µM dNTP, 2.5 U Pwo DNA Polymerase, 1X Pwo PCR buffer with 2 mM MgSO4, and 2 external primers at a concentration of 1 µM. The PCR program consisted of 10 cycles at 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 60 seconds. These variegated sequences were digested and cloned into the V3-23 framework instead of the CDR1 / 2 filler.

Approximately 7 X 107 independent transformants were obtained. In this diversity, diversity of CDR3 can be cloned from donor populations or synthetic DNA.

In addition, this application describes the following sections:

1. A method for cleaving single stranded nucleic acid sequences at a desired location, the method comprising the steps of:

(i) contacting the nucleic acid with a single stranded oligonucleotide, the single stranded oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired, and including a sequence that with its complement in the nucleic acid forms a site of recognition

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of restriction endonuclease which with the restriction results in nucleic acid cleavage at the desired location; Y

(ii) cleaving the nucleic acid only at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that Excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature.

2. A method for cleaving single stranded nucleic acid sequences at a desired location, the method comprising the steps of:

(i)

contacting the nucleic acid with a partially double stranded oligonucleotide, the single stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired, and the double stranded region of the oligonucleotide having a restriction endonuclease recognition site of Type II-S, whose cleavage site is located at a known distance from the recognition site; Y

(ii)

cleaving the nucleic acid only at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single stranded region of the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that Excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature.

3. In a method for presenting a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively presenting at least a part of the family's diversity, the improvement being characterized because the at least a part of presented peptide, polypeptide or protein is encoded at least in part by a nucleic acid that has been cleaved at a desired location by a method comprising the steps of:

(i)

contacting the nucleic acid with a single stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired, and including a sequence that with its complement in the nucleic acid forms an endonuclease recognition site of restriction that with the restriction results in nucleic acid cleavage at the desired location; Y

(ii)

cleaving the nucleic acid only at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that Excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature.

4. In a method for presenting a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively presenting at least part of the family's diversity, the improvement being characterized because the peptide, polypeptide or protein presented is encoded by a DNA sequence comprising a nucleic acid that has been cleaved at a desired location

(i)

contacting the nucleic acid with a partially double-stranded oligonucleotide, the single stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired, and the double stranded region of the oligonucleotide having a restriction endonuclease recognition site of Type II-S, whose cleavage site is located at a known distance from the recognition site; Y

(ii)

cleaving the nucleic acid only at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single stranded region of the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region sufficiently large to allow the two strands to associate

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so that excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature.

5. A method for presenting a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively presenting at least a part of the family's diversity, the method comprising the steps of:

(i)

prepare a collection of nucleic acids that at least partially encode members of the diverse family,

(ii)

make single strands to nucleic acids;

(iii) cleaving the single stranded nucleic acids at a desired location by a method comprising the steps of:

(to)

contacting the nucleic acid with a single stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site which with the restriction results in nucleic acid cleavage at the desired location, and

(b)

cleaving the nucleic acid only at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature; Y

(iv) presenting a member of the family of peptides, polypeptides or proteins encoded, at least in part, by the nucleic acids cleaved on the surface of the genetic package and collectively presenting at least a portion of the family's diversity.

6. A method of presenting a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively presenting at least a portion of the family's diversity, the method comprising the steps of:

(i)

prepare a collection of nucleic acids that encode at least in part, members of the diverse family,

(ii)

make single strands to nucleic acids;

(iii) cleaving the single stranded nucleic acids at a desired location by a method comprising the steps of:

(to)

contacting the nucleic acid with a single stranded oligonucleotide, the single stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired, and the double stranded region of the oligonucleotide having a Type restriction endonuclease recognition site II-S, whose cleavage site is located at a known distance from the recognition site; Y

(b)

cleaving the nucleic acid only at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single stranded region of the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature; Y

(iv) presenting a member of the family of peptides, polypeptides or proteins encoded, at least in part, by the nucleic acids cleaved on the surface of the genetic package and collectively presenting at least a portion of the family's diversity.

7. A library comprising a collection of genetic packages that presents a member of a diverse family of peptides, polypeptides or proteins and collectively presents at least a portion of the diversity of the family, the library being produced using the methods of the sections 3, 4, 5 or 6.

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8. A library comprising a collection of genetic packages that presents a member of a diverse family of peptides, polypeptides or proteins and that collectively presents at least a portion of the family, the peptides, polypeptides or proteins presented by sequences being encoded of DNA comprising at least partly sequences produced by cleaving single stranded nucleic acid sequences at a desired location by a method comprising the steps of:

(i)

contacting the nucleic acid with a single stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired, and including a sequence that with its complement in the nucleic acid forms an endonuclease recognition site of restriction that with the restriction results in the cleavage of the nucleic acid at the desired location; Y

(ii)

cleaving the nucleic acid only at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that Excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature.

9. A library comprising a collection of genetic packages that presents a member of a diverse family of peptides, polypeptides or proteins and that collectively presents at least a portion of the diversity of the family of the presented peptides, polypeptides or proteins that It is encoded by DNA sequences that comprise at least partly sequences produced by cleaving single stranded nucleic acid sequences at a desired location by a method comprising the steps of:

(i)

contacting the nucleic acid with a partially double stranded oligonucleotide, the single stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which the cleavage is desired, and the double stranded region of the oligonucleotide having a restriction endonuclease recognition site of Type II-S, whose cleavage site is located at a known distance from the recognition site where nucleic acid cleavage is desired; Y

(ii)

cleaving the nucleic acid only at the Type II-S cleavage site formed by the complementation of the nucleic acid and the single stranded region of the oligonucleotide;

the contacting and cleavage steps being carried out at a temperature sufficient to maintain the nucleic acid in substantially single stranded form, the oligonucleotide being functionally complementary to the nucleic acid along a region large enough to allow the two strands to associate so that Excision can occur at the chosen temperature and at the desired location, and excision being carried out using a restriction endonuclease that is active at the chosen temperature.

10.

The methods according to any one of sections 1 to 9, wherein the nucleic acids encode at least a portion of an immunoglobulin.

eleven.

The methods according to section 10, wherein the immunoglobulin comprises a single chain Fab or Fv.

12.

The methods according to section 10 or 11, wherein the immunoglobulin comprises at least a portion of a heavy chain.

13.

The methods according to section 12, in which at least a portion of the heavy chain is human.

14.

The methods according to section 10 or 11, wherein the immunoglobulin comprises at least a portion of FR1.

fifteen.

The methods according to section 14, in which at least a portion of the FR1 is human.

16.

The methods according to section 10 or 11, wherein the immunoglobulin comprises at least a portion of a light chain.

17.

The methods according to section 16, in which at least a portion of the light chain is human.

18.

The methods according to any one of sections 1 to 9, in which the nucleic acid sequences derive at least in part from patients suffering from at least one autoimmune disease and / or cancer.

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19.

The methods according to section 18, in which the autoimmune disease is selected from the group comprising lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome or vasculitis.

twenty.

The methods according to section 18, wherein the nucleic acids are isolated at least in part from the group comprising peripheral blood cells, bone marrow cells, spleen cells or lymph node cells.

twenty-one.

The methods according to section 5 or 6, further comprising a stage of nucleic acid amplification between stages (i) and (ii), between stages (ii) and (iii) or between stages (iii) and (iv).

22

The methods according to section 21, in which the amplification stage uses geneRACE ™.

2. 3.

The methods according to any one of sections 1 to 9, wherein the temperature is between 45 ° C and 75 ° C.

24.

The methods according to section 23, in which the temperature is between 50 ° C and 60 ° C.

25.

The methods according to section 24, in which the temperature is between 55 ° C and 60 ° C.

26.

The methods according to section 1, 3, 5 or 8, in which the length of the single stranded oligonucleotide is between 17 and 30 bases.

27.

The methods according to section 26, in which the length of the single stranded oligonucleotide is between 18 and 24 bases.

28.

The methods according to section 1, 3, 5 or 8, in which the restriction endonuclease is selected from the group comprising: MaeIII, Tsp45I, HphI, BsaJI, AluI, BlpI, DdeI, BglII, MslI, BsiEI, EaeI , EagI, HaeIlI, Bst4CI, HpyCH4III, HinfI, MlyI, PleI, MnlI, HpyCH4V, BsmAI, BpmI, XmnI, or SacI.

29.

The methods according to section 28, wherein the restriction endonuclease is selected from the group comprising Bst4CI, TaaI, HpyCH4III, BlpI, HpyCH4V or MslI.

30

The methods according to section 2, 4, 6 or 9, in which the length of the single stranded region of the partially double stranded oligonucleotide is between 14 and 22 bases.

31.

The methods according to section 30, in which the length of the single stranded region of the partially double stranded oligonucleotide is between 14 and 17 bases.

32

The methods according to section 31, in which the length of the single stranded region of the oligonucleotide is between 18 and 20 bases.

33.

The methods according to section 2, 4, 6 or 9, in which the length of the double stranded region of the partially double stranded oligonucleotide is between 10 and 14 base pairs formed by a stem and its palindrome.

3. 4.

The methods according to section 33, wherein the partially double stranded oligonucleotide comprises a 3 to 8 base loop between the stem and the palindrome.

35

The methods according to sections 2, 4, 6 or 9, in which the Type II-S restriction endonuclease is selected from the group comprising AarICAC, AceIII, Ebr7I, BbvI, BbvII, Bce83I, BceAI, BcefI, BciVI , BfiI, BinI, BscAI, BseRI, BsmFI, BspMI, EciI, Eco57I, FauI, FokI, GsuI, HgaI, HphI, MboII, MlyI, MmeI, MnlI, PleI, RleAI, SfaNI, SspD5I, Sth132I, Sth132I, Tth132q, Sth132I, Tth132I, Tth132q , or UbaPI.

36.

The methods according to section 35, in which the Type II-S restriction endonuclease is Fokl.

37.

A method for preparing single stranded nucleic acids for cloning into a vector, the method comprising the steps of:

(i)

contacting a single stranded nucleic acid sequence that has been cleaved with a restriction endonuclease with a partially double stranded oligonucleotide, the single stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region that remains after cleavage, including the double stranded region of the oligonucleotide any sequences necessary to return the sequences that remain after cleavage to the appropriate and original reading frame for expression and containing a 5 'restriction endonuclease recognition site of those sequences; Y

(ii)

cleaving the partially double stranded oligonucleotide sequence only at the restriction endonuclease recognition site contained within the double stranded region of the partially double stranded oligonucleotide.

Claims (1)

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